Receptor tyrosine kinases (RTK) can be oncogenic drivers in cancer due to genetic aberrations such as amplification, mutations or fusion events or via overexpression (M. A. Lemmon, K. M. Ferguson, Cell 130, 213 (2007)). Most aberrations in RTK result in ligand-independent activation of the receptor and activation of downstream signalling promoting cell growth and proliferation and increased survival. The class III RTK including KIT, platelet-derived growth factor receptor (PDGFR) alpha and beta, colony-stimulating factor 1 receptor (CSF1R), and the Fms-like tyrosine kinase 3 receptor (FLT3) is implicated in a variety of human cancers (K. Verstraete. S. N. Savvides, Nat. Rev. Cancer 12, 753 (2012)).
The gene encoding KIT is located on Chr 4 and comprises 21 exons (J. Lennartsson, L. Ronnstrand, Physiol Rev. 92, 1619 (2012)). The 976 amino acids of the KIT protein are divided into key domains: an extracellular domain, a transmembrane domain, a juxtamembrane domain (JM) and kinase domain separated by a kinase insert (KID) in the middle. The mature protein is ˜145 KDa following N glycosylation and is expressed at the cell surface. Following stem cell factor (SCF) binding, the dimerisation increases the intrinsic kinase activity phosphorylating tyrosine residues in the JM domain (Y547, Y553, Y568 and Y570) followed by phosphorylations in the KID (Y703, Y721, Y729/730) and finally the activation loop (Y823) (J. P. DiNitto et al., J. Biochem. 147, 601 (2010)). Some phosphoylations sites on KIT are key docking sites for adaptors and downstream effectors propagating the activation signal. PI3K, Src and MAPK are key signalling pathways activated downstream of KIT. Regulation of KIT signalling includes internalization and subsequent degradation of the receptor, phosphorylation of Ser 741 and 746, and dephosphorylation of tyrosine residues by phosphatases such as SHP1.
KIT-driven signaling plays a key role in specific cell types, including interstitial cells of Cajal (ICCs), melanocytes, mast cells, germ cells and some hematopoietic stem cells (J. Lennartsson, L. Ronnstrand, Physiol Rev. 92, 1619 (2012)). Aberrations of KIT are observed in malignancies derived from these cell types. For example, KIT mutations are reported in gastrointestinal stromal tumours (originating from ICC), in mastocytosis and in melanomas.
Mutations in KIT in cancer affect multiple exons with hotspot mutations observed in the JM and kinase domains (J. Lennartsson, L. Ronnstrand, Physiol Rev. 92, 1619 (2012)). Mutations in the JM domain are thought to remove the autoinhibitory interaction of the JM domain with the kinase domain (J. P. DiNitto et al., J. Biochem. 147, 601 (2010)). Lower frequency mutations are present in exon 9 (extracellular Ig domain 5) and 13 (ATP binding pocket and gatekeeper). Mutations in the JM domain are observed in GIST while mutations affecting the kinase domain, in particular the A loop are frequently observed in mastocytosis. Similarly, PDGFR mutations in GIST affect both the JM domain and the kinase domain (C. Bahlawane et al., Cell Commun. Signal. 13, 21 (2015)).
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract (C. M. Barnett, C. L. Corless, M. C. Heinrich. Hematol. Oncol. Clin. North Am. 27, 871 (2013)). GISTs are most commonly found in the stomach and small intestine. Neoplastic GIST originate from the same precursor cells as the ICC and the vast majority of GIST express KIT protein initially called CD117. KIT mutations affecting exon 11 were first identified in GIST in 1998 (S. Hirota et al., Science 279, 577 (1998)). The same publication also reported the oncogenicity of KIT mutations expressed ectopically in Ba/F3 cells and their constitutive kinase activation. 75-80% GIST harbor KIT mutations and ˜10% PDGFR mutations (J. A. Fletcher, Cancer Res. 76, 6140 (2016)). Rare aberrations in BRAF, NF1 and SDH account for what is referred to as WT KIT (C. M. Barnett, C. L. Corless, M. C. Heinrich, Hematol. Oncol. Clin. North Am. 27, 871 (2013)).
Imatinib was the first KIT inhibitor tested in GIST, demonstrating remarkable activity in patients with advanced GIST (G. D. Demetri et al., N. Engl. J. Med. 347, 472 (2002), J. Verweij et al., Lancet 364, 1127 (2004), C. D. Blanke et al., J. Clin. Oncol. 26, 626 (2008)). A meta-analysis of 2 large clinical studies concluded that patients with exon 9 mutations in KIT or other mutations had worse prognosis than patients with exon 11 mutations (Metagist, J. Clin. Oncol. 28, 1247 (2010)). In addition, a high dose imatinib (800 mg) did not improve progression-free survival in patients with exon 9 mutations compared to the standard dose (400 mg). Clinical resistance to imatinib was first reported in 2005 (C. R. Antonescu et al., Clin. Cancer Res. 11, 4182 (2005)) but a larger study following patients treated with imatinib as part of a PhII study B2222 showed a reactivation of KIT and KIT signalling with patients who have initially benefited from imatinib relapsed (M. C. Heinrich et al., J. Clin. Oncol. 24, 4764 (2006)). Secondary resistance mutations were noted at key residues: V654A in the ATP-binding pocket, T670I at the gatekeeper residue and A loop (D816X, D820X, N822K, Y823D). In addition, so called “primary resistance” to imatinib was mainly observed in patients with exon9 mutations. Overall, 50% of patients developed resistance within 2 years (C. D. Blanke et al., J. Clin. Oncol. 26, 626 (2008).).
Sunitinib is a multikinase inhibitor including KIT and PDGFR. Sunitinib demonstrated clinical activity in GIST patients following progression on imatinib (G. D. Demetri et al., Lancet 368, 1329 (2006)). Clinical benefit with sunitinib was observed in patients with primary exon 9 mutations. In addition, patients with secondary mutations affecting exon 13 and 14 had longer progression-free and overall survival compared to patients with secondary mutations affecting the A loop (M. C. Heinrich et al., J. Clin. Oncol. 26, 5352 (2008)). Clinical progression with sunitnib was observed within 1 year of treatment. Ectopic expression of KIT with primary and secondary mutations in CHO cells showed that sunitinib reduced KIT phosphorylation preferentially when KIT aberrations affected the ATP binding pocket or the gatekeeper.
Regorafenib, another multikinase inhibitor has shown clinical activity in patients with GIST after relapse to imatinib and sunitinib (G. D. Demetri et al., Lancet 381, 295 (2013)). The PhIII study reported a median PFS of 4.8 months.
Accordingly, there is a need for KIT inhibitors that inhibit secondary KIT mutations, and furthermore, are selective against KDR, particularly as existing treatments are ineffective against such secondary mutations. There is also a need for KIT inhibitors that inhibit primary KIT mutations and wildtype KIT.